hKSR-2, a vitamin D-regulated gene, inhibits apoptosis in arabinocytosine-treated HL60 leukemia cells.

Ras signaling can be modulated by the scaffolding activity of kinase suppressor of Ras-1 (KSR-1) and by the hKSR-2 protein, resulting in diverse phenotypic outcomes. The mitogen-activated protein kinase cascade downstream from Ras and KSRs includes Raf-1 and extracellular signal-regulated kinase 1/2 kinases, known to enhance survival potential of a range of cell types. Because the molecular events that increase survival of HL60 cells induced to differentiate toward monocytic phenotype by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are not known, we investigated if KSR proteins provide a survival function in these cells. We found that whereas kinase suppressor of Ras-1 had no detectable effect on cell survival in the system studied here, 1,25-(OH)2D3-induced up-regulation of hKSR-2 enhanced the resistance of HL60 cells to arabinocytosine. Knockdown of hKSR-2 by either small interfering RNA or antisense oligonucleotides increased arabinocytosine-induced apoptosis, which was accompanied by reduced Bcl-2/Bax and Bcl-2/Bad ratios, and increased caspase-3 activating cleavage. In contrast, up-regulation of Mcl-1 was not abrogated by anti-sense (AS) AS-hKSR-2, pointing to a specific role of Bcl-2 in control of 1,25-(OH)2D3-induced increased cell survival. These findings are consistent with the previously shown lack of fully differentiated monocytic cells in HL60 cultures exposed to 1,25-(OH)2D3 in which hKSR-2 was knocked down, suggesting that optimal differentiation of these cells requires enhanced antiapoptotic mechanisms provided, at least in part, by hKSR-2. Collectively, these results suggest that hKSR-2 may offer a new target for novel therapies of acute myelogenous leukemia.